Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr

Cell Wall Antibiotics Provoke Accumulation of Anchored mCherry in the Cross Wall of Staphylococcus aureus

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK motif SPsasF. mCh-hybrids supplied with the +YSIRK motif SPlip were always expressed higher than those with −YSIRK motif SPsasF. To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall

Simplivariate Models: Uncovering the Underlying Biology in Functional Genomics Data

One of the first steps in analyzing high-dimensional functional genomics data is an exploratory analysis of such data. Cluster Analysis and Principal Component Analysis are then usually the method of choice. Despite their versatility they also have a severe drawback: they do not always generate simple and interpretable solutions. On the basis of the observation that functional genomics data often contain both informative and non-informative variation, we propose a method that finds sets of variables containing informative variation. This informative variation is subsequently expressed in easily interpretable simplivariate components

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor

ACTION DE LA FLORE MICROBIENNE DU TRACTUS DIGESTIF SUR LE MÉTABOLISME DES SELS BILIAIRES. DÉGRADATION DE L'ACIDE β-MURICHOLIQUE

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INFLUENCE D'UNE SUPPLÉMENTATION DE LA RATION EN SELS BILIAIRES CONJUGUÉS SUR L'ÉQUIPEMENT EN ACIDES BILIAIRES DE L'INTESTIN GRÊLE DU RAT. MODIFICATIONS DE L'ABSORPTION APPARENTE DES ACIDES GRAS

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Structural and dynamic characterization of the C-terminal tail of ErbB2: disordered but not random

International audienceErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers, associated with poorprognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in thelast twenty years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding ofthe receptor function and targeted inhibition. However, ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains verypoorly characterized in terms of structure, dynamics and detailed functional mechanism. Yet, it is where signal transduction istriggered, via phosphorylation of tyrosine residues, and carried out, via interaction with adaptor proteins. Here we report the firstdescription of ErbB2 disordered tail at atomic resolution using NMR, complemented by SAXS. We show that although no part ofCtErbB2 has any fully populated secondary or tertiary structure, it contains several transient helices and numerous transientPPII helices, populated up to 20 and 40% respectively, and low but significant compaction. The presence of some structuralelements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2’sauto-regulation processes. In addition, the transient formation of PPII helices is compliant with previously suggested interactionswith SH3 domains. All in all, our in-depth structural study opens new perspectives in the mechanistic understanding of ErbB2